1. Field of the Invention
This invention relates to peptides, compositions and methods involving these peptides for the inhibition of membrane fusion by paramyxoviruses and, in particular, membrane fusion mediated by Hendra virus and Nipah virus.
2. Description of the Background
Membrane fusion is a ubiquitous cell biological process. Fusion events which mediate cellular housekeeping functions, such as endocytosis, constitutive secretion, and recycling of membrane components, occur continuously in all eukaryotic cells.
Additional fusion events occur in specialized cells. Intracellularly, for example, fusion events are involved in such processes as occur in regulated exocytosis of hormones, enzymes and neurotransmitters. Intercellularly, such fusion events feature prominently in, for example, sperm-egg fusion and myoblast fusion.
Fusion events are also associated with disease states. For example, fusion events are involved in the formation of giant cells during inflammatory reactions, and particularly, the entry of all enveloped viruses into cells.
For example, the paramyxoviruses are negative-stranded RNA containing enveloped viruses encompassing a variety of important human and animal pathogens including measles virus (MeV), canine distemper virus (CDV), human parainfluenza viruses (hPIV) 1-4, and simian virus 5 (SV5) (reviewed in (Lamb and Kolakofsky, 1996)). These viruses contain two principal membrane-anchored glycoproteins which appear as spikes projecting from the envelope membrane of the viral particle when imaged in the electron microscope. One glycoprotein is associated with virion attachment to the host cell and, depending on the particular paramyxovirus, has been designated as either the hemagglutinin-neuraminidase protein (FIN), the hemagglutinin protein (H), or the G protein which has neither hemagglutinating nor neuraminidase activities. The attachment glycoproteins of the paramyxoviruses are type II integral membrane proteins. In several cases where the attachment glycoprotein is of the HN type, it is sialic acid moieties which serve as receptors for virus entry. For paramyxoviruses possessing an H or G attachment glycoprotein the identity of host cell receptors are not known, except in the case of MeV where CD46 can serve as a functional receptor (Naniche et al., 1993). The second glycoprotein is the fusion protein (F), a type I membrane glycoprotein, which facilitates the membrane fusion event between the virion and host cell during virus infection (reviewed in Lamb, 1993).
Fusion of the membrane of enveloped viruses with the plasma membrane of a receptive host cell is a prerequisite for viral entry and infection and an essential step in the life cycle of all enveloped viruses. Following paramyxovirus attachment to a permissive host cell, fusion at neutral pH between the virion and plasma membranes ensues, resulting in delivery of the nucleocapsid into the cytoplasm (reviewed in (Lamb and Kolakofsky, 1996)). In a related process, cells expressing these viral glycoproteins at their surfaces can fuse with receptor-bearing cells, resulting in the formation of multinucleated giant cells (syncytia). The paramyxovirus F glycoprotein shares several features with other viral membrane fusion proteins, including the envelope glycoprotein of retroviruses like gp120/gp41 of HIV-1, and hemagglutinin (HA) of influenza virus (reviewed in (Hernandez et al., 1996)). The biologically active F protein consists of two disulfide linked subunits, F and F2, that are generated by the proteolytic cleavage of a precursor polypeptide known as Fo (reviewed in (Klenk and Garten, 1994; Scheid and Choppin, 1974)). In all cases the membrane-anchored subunit contains a new amino terminus that is hydrophobic and highly conserved across virus families and referred to as the fusion peptide (reviewed in (Hunter, 1997)). The fusion peptide is an important structural element required to mediate virion/host cell membrane fusion. All paramyxoviruses studied to date, with the exception of SV5, under certain circumstances, require both the attachment and F glycoprotein for membrane fusion (Paterson, Johnson, and Lamb, 1997). Although evidence of physical interactions have only been rarely detected, it is hypothesized that the binding protein must somehow signal and induce a conformational change in F leading to virion/host cell membrane fusion (Lamb, 1993).
In 1994, a new paramyxovirus, now called Hendra virus (HeV) and recognized to be a member of the subfamily Paramyxovirinae, was isolated from fatal cases of respiratory disease in horses and humans, and was shown to be distantly related to MeV and other members of the morbillivirus genus (Murray et al., 1995). The first outbreak of severe respiratory disease in the Brisbane suburb of Hendra resulted in the death of 13 horses and their trainer, and the non-fatal infection of a stable hand and a further 7 horses. At approximately the same time, in an unrelated incident almost 100 km north of Hendra, a 35-year-old man experienced a brief aseptic meningitic illness after caring for and assisting at the necropsies of two horses subsequently shown to have died as a result of HeV infection. Thirteen months later the man suffered severe encephalitis characterized by uncontrolled focal and generalized epileptic-activity. A variety of studies that were performed in the evaluation of this fatality, including serology, PCR, electron microscopy and immunohistochemistry, strongly suggested that HeV was indeed the cause of this patient's encephalitis, and the virus was acquired from the HeV-infected horses (O'Sullivan et al., 1997). In all, fifteen horses and two people died in the two episodes. At the time the source of the emerging virus was undetermined, but more recently it has been found that approximately 50% of Australian fruit bats, commonly known as flying foxes, have antibodies to HeV and HeV-like viruses have been isolated from bat uterine fluids and it appears that these animals are the natural host for the virus (Field et al., 2001; Halpin et al., 1999; Halpin et al., 2000; Young et al., 1996).
More recently, the nucleic acid sequence of the genes of HeV has been compared with those of other paramyxoviruses (Wang et al., 1998; Yu et al., 1998a; Yu et al., 1998b). These later studies have confirmed that HeV is a member of the Paramyxoviridae, subfamily Paramyxovirinae, but low homology with other subfamily members and the presence of several novel biological and molecular features such as F protein cleavage at a single lysine residue and genome length suggest classification in a new genus within the Paramyxovirinae.
Subsequent to these events, an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore occurred in 1998 (Anonymous, 1999). The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 cases of encephalitis, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. This may represent a near 40% fatality rate upon infection, because the incidence of subclinical human infections during these episodes has not been well defined. Electron microscopic, serologic, and genetic studies have since indicated that this virus also belongs to the Paramyxovirinae subfamily, and was most closely related to HeV. This virus was named Nipah virus (NiV) after the small town in Malaysia from which the first isolate was obtained from the cerebrospinal fluid of a fatal human case (Chua et al., 2000; Chua et al., 1999; Goh et al., 2000; Lee et al., 1999; Lim et al., 2000). NiV and HeV are now recognized as the prototypic members of a new genus within the Paramyxovirinae subfamily called Henipavirus (Wang et al., 2001; Wang and Eaton, 2001). Both HeV and NiV are unusual among the paramyxoviruses in their ability to infect and cause potentially fatal disease in a number of host species, including humans and in that they have an exceptionally large genome.
These viruses classified as Biosafety Level 4 agents (BSL-4) and have the potential to be used as biological warfare agents. There are no existing antiviral therapies effective against these viruses, and the only therapies in existence to any viruses in the paramyxovirus family are attenuated vaccines for the prevention of infection by MeV and Mumps virus. Accordingly, agents and compositions effective against infections by viruses in the paramyxovirus family are particularly desirable. In this connection, the heptad peptides of the instant invention represent an effective therapy for paramyxovirus infection resulting from, e.g., a biological weapon, a natural outbreak, or a BSL-4 laboratory accident.